The 10-member sarcomeric myosin heavy chain gene family has been studied extensively and mutations in half of its members have been implicated in disease. Myosin motors convert chemical energy into mechanical force by amplifying the ATP-driven conformational rotation of myosin?s lever arm, which consists of a helical pliant region and the ?-helix of the heavy chain stabilized and stiffened by essential and regulatory light chains; hereafter referred to as the lever arm. While a great deal of study has been devoted to the catalytic domain and converter, the lever arm has often been treated as simply a semi-rigid extension of the converter to amplify the stroke size of the motor. For a given isoform of myosin the entire lever arm is highly conserved across species, but it is highly variant amongst the 10 isoforms, suggesting that the sequences of lever arm ?- helices confer specific functions. Given the high sequence conservation of the ?-cardiac lever arm across species and the high density of pathogenic mutations in it, we hypothesize that this region is an important regulatory domain that modulates myosin function and testing that hypothesis is the focus of this proposal. We propose an interdisciplinary collaboration among the Spudich, Perkins and Leinwand laboratories to study the effects of disease-causing mutations in the lever arm by integrating the biophysical characterization of isolated lever arms and myosin motor functional assays with cardiac cell biology. In the previous grant period, the Leinwand, Spudich and Geeves laboratories produced and characterized a number of disease-causing mutations of the human ?-cardiac myosin motor for their biochemical and kinetic properties, but none of these studies included lever arm mutations. Because of clinical hypercontractility of hypertrophic cardiomyopathy (HCM) patients and hypocontractility of dilated cardiomyopathy (DCM) patients, we hypothesize that HCM mutations will most likely increase the stiffness of the lever arm, whereas, DCM mutations will cause the lever arm to be less stiff. In Aim I, we will determine the biochemical and mechanical properties of the lever arm of WT ?-myosin using atomic force microscopy (AFM). AFM has emerged as a powerful tool for investigating the elasticity of proteins in addition to probing their folding/unfolding dynamics. Until now, AFM technology did not have the resolution to study the mechanics of the 9-nm long lever arm. However, the Perkins lab?s recent advances in single-molecule AFM techniques will enable us to compare the mechanical properties of the WT -cardiac myosin lever arm ?-helix to ones carrying cardiomyopathy-causing mutations. In Aim II, we will measure the impact of the lever arm mutations on in vitro subfragment-1 (S1) motor function using ATPase, gliding filament and optical tweezer assays. Finally, in Aim III we will integrate these biophysical and biomechanical findings into cells by introducing WT and lever arm mutant full length ?-cardiac myosins into cardiac myocytes and determining their effects on sarcomere integrity, sarcomere dynamics and contractility.